Field of the Invention
The invention concerns an oxygen-independent cholesterol-converting enzyme, a process for its isolation from particular microorganisms as well as the use of the enzyme to determine cholesterol.
The quantitative determination of cholesterol in blood with the aid of enzymatic tests has for a long time been a proven method in clinical chemistry (Flegg 1973, Richmond 1973). A cholesterol oxidase is usually used as the enzyme which catalyses the oxidation of cholesterol (5-cholesten-3-.beta.-ol) to 4-cholesten-3-one and H.sub.2 O.sub.2. ##EQU1##
In this case oxygen serves as an electron acceptor. The provision of this enzyme on an industrial scale from microorganisms of the Schizophyllum, Streptoverticillium, Brevibacterium, Nocardia, Rhodococcus or streptomyces classes (Noma & Nakayama 1976, EP 0560 983; Liu et al. 1980, Ishizaki 1989, Halpern 1981, Aihara et al. 1986, Fujishiro et al. 1990, EP 0452 112) is established.
However, the oxygen dependency of cholesterol oxidase is a major disadvantage and the main reason for test inaccuracies. It often causes problems in calibrating the test since the dependency on oxygen partial pressure results in an altitude dependency as well as in a temperature dependency of the test.
In addition the quantitative determination of cholesterol from blood with the aid of cholesterol oxidases by means of a coupled colour reaction is susceptible to interference due to the H.sub.2 O.sub.2 that is formed: H.sub.2 O.sub.2 is removed from the reaction equilibrium due to its high reactivity with for example bilirubin or drugs.
An oxygen-independent cholesterol-converting enzyme, i.e. a NAD- or NADP-dependent cholesterol dehydrogenase which is obtainable from an anaerobic microorganism (Eubacterium sp.) or from liver tissue of warm-blooded animals, is described in DE 2649749. The major disadvantage of the enzyme described in this application is that the enzyme also has to be isolated under an inert gas atmosphere. A scale-up of the process and the provision of the said enzyme on a larger scale is not feasible. Moreover, the enzyme requires NAD(P) as a cofactor so that an additional reaction step (enzymatic or chemical) is necessary for colour formation. This usually results in an increase in costs and a higher susceptibility to interference. Appropriate NAD(P)-independent dehydrogenases have been previously known only for other substrates such as for example glucose or glycerol (Duine 1991, Ameyama et al. 1985, Ameyama 1982, EP 0354 441, Ameyama et al. 1981, EP 0 120 440).